Somatic hypermutation of immunoglobulin genes occurs at a frequency that is a million times greater than mutation in other genes. Mutations are found in both variable genes and switch regions before constant genes. The molecular mechanism that introduces these mutations is intensely being studied. Hypermutation is initiated when the activation-induced cytidine deaminase (AID) protein deaminates cytosine in DNA to uracil, which causes C:G mutations. However, in B lymphocytes, substitutions of all four bases occur at similar levels, indicating that other proteins are required to generate mutations of A:T base pairs. We are studying how DNA polymerases are involved in the process. Polymerase eta is clearly involved in the process by causing substitutions of A:T basepairs, whereas polymerase iota has a controversial role. To examine polymerase iota in a genetically defined background, we backcrossed the 129 nonsense mutation to the C57BL/6 strain for six generations. Class switch recombination and hypermutation were studied in these mice and in congenic mice doubly deficient for both polymerases eta and iota. The absence of both polymerases did not affect production of IgG1, indicating that these enzymes are not involved in switch recombination. Poli-/-F6 mice had the same types of nucleotide substitutions in variable genes as their C57BL/6 counterparts, and mice doubly deficient for polymerases eta and iota had the same mutational spectrum as Polh-/- mice. Thus, polymerase iota did not contribute to the mutational spectra, even in the absence of polymerase eta.